Vascular mimicry as a facilitator of melanoma brain metastasis

Melanoma has the highest propensity among solid tumors to metastasize to the brain. Melanoma brain metastases (MBM) are a leading cause of death in melanoma and affect 40–60% of patients with late-stage disease. Therefore, uncovering the molecular mechanisms behind MBM is necessary to enhance therapeutic interventions. Vascular mimicry (VM) is a form of neovascularization linked to invasion, increased risk of metastasis, and poor prognosis in many tumor types, but its significance in MBM remains poorly understood. We found that VM density is elevated in MBM compared to paired extracranial specimens and is associated with tumor volume and CNS edema. In addition, our studies indicate a relevant role of YAP and TAZ, two transcriptional co-factors scarcely studied in melanoma, in tumor cell-vasculogenesis and in brain metastasis. We recently demonstrated activation of the Hippo tumor suppressor pathway and increased degradation of its downstream targets YAP and TAZ in a metastasis impaired cell line model. In the current study we establish the utility of anti-YAP/TAZ therapy in mouse models of metastatic melanoma whereby treatment effectively inhibits VM and prolongs survival of mice with MBM. The data presented herein suggest that VM may be an important and targetable mechanism in melanoma and that VM inhibition might be useful for treating MBM, an area of high unmet clinical need, thus having important implications for future treatment regimens for these patients. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05217-z.

The TMAs were scanned using a multispectral Vectra Polaris instrument (Akoya Biosciences).
Then, images were analyzed using the AQUA method of Quantitative immunofluorescence (QIF) (Navigate Biopharma, Carlsbad, California, USA).This analysis enabled objective and sensitive measurement of targets within user-defined tissue compartments (Camp et al, 2002).Briefly, the QIF score of YAP marker in all cells (DAPI+), the S100-positive tumor-cell compartment and the surrounding S100-negative non-tumor/stromal-cell tissue area was calculated by dividing the target pixel intensities by the area of S100-positive or S100-negative pixels.This allows for comparisons across cases with dissimilar tumor and stromal content.The fluorescence scores obtained are normalized by the exposure time and bit depth, making them comparable across cases.Methods are also described previously [3].

Conditioned medium vascular mimicry
Cl.2A, YUSIK, and YUVENA cells were plated in a 10-cm dish and were serum starved for 24 hours.Conditioned medium was collected the day of the VM assay.100µl chilled Matrigel (Corning) was added to a chilled 48-well plate and incubated for at least 30 minutes at 37 o C to allow Matrigel polymerization.30,000 YUKOLI, YUKSI or YUCOT melanoma cells in 300µL were seeded dropwise to each well in reduced serum media as the control or conditioned medium from Cl.2A, YUSIK or YUVENA.Images were taken 4 hours later and analyzed.
Vascular mimicry with endothelial cell co-culture MS1 and bEnd.3 cells were plated in full culture medium in a 0.4µM pore insert fit for a 24-well plate and allowed grow overnight.200µl chilled Matrigel (Corning) was added to the empty wells of the 24-well plate and incubated for at least 30 minutes at 37 o C to allow Matrigel polymerization.0.4µM pore inserts were moved to the wells with the Matrigel and 60,000 B16 or B16.F10 cells in 600µM reduced serum media were plated dropwise on the Matrigel.VM was imaged and analyzed after 24 hours.

Vascular mimicry with Lenvatinib and anti-VEGF treatment
Vascular mimicry assay was completed as described previously [4].100µl chilled Matrigel (Corning or R&D) was added to a chilled 48-well plate and incubated for at least 30 minutes at 37 o C to allow Matrigel polymerization.30,000 melanoma cells in 300µL were seeded dropwise to each well in 2% serum culture media apart from the YUMM lines being seeded in full media.
Lenvatinib, mouse anti-VEGF, or human anti-VEGF (bevacizumab) were added to the mixture of cells prior to adding dropwise to the well.The final concentration of lenvatinib (LC Laboratories) used for treatment was 10µM and anti-VEGF was 1mg/mL.Mouse anti-VEGF was purchased from Absolute Antibody (clone G6-31, Cat# 1022-2.0)and human anti-VEGF (bevacizumab; MVASI) was purchased from Amgen (NDC SS513-206-01).

Endothelial tube formation assay
The in vitro angiogenesis assay kit (ECM640-Millipore) was used.Briefly, 50 µl of chilled ECMatrix™ Gel Solution was added to multiple wells of a chilled 96-well plate and incubated at 37°C for at least 30 min to allow polymerization.Normal endothelial cells (NEC) and tumor endothelial cells (TEC) were trypsinized and counted, and 6,000 cells in serum-free endothelial cell medium were added to each well.Tube formation was quantified by the ImageJ angiogenic analyzer.b.End5 was used as the TEC.b.End5 is an endothelial cell line isolated from brain tissue of a Balb/c mouse with endothelioma.This was a gift from the depositor (Culture Collection.org.uk,Cat# 96091930) to Dr. Rafael Reuten at the University of Copenhagen.NEC is a normal endothelial cell derived from a healthy brain of a Balb/C mouse (Cell Biologics, Cat# BALB-5023).

Western Blot
For western blots, cells were lysed using RIPA solution supplemented with 1mM Na3VO4, 1mM PMSF, and 1M NAF containing the protease inhibitor cocktail.Protein concentrations of lysates was determined Bicinchoninic Acid (BCA) assay.20-30 µg of protein was diluted in a sample buffer (4X Laemmli Buffer: Bio-Rad supplemented with 1part β-mercaptoethanol) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Detection of proteins was done with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (#7076 and #7074 respectively: Cell Signaling).Antibodies used in this study are in the table below: Quantitative RT-PCR Quantitative RT-PCR was performed as described previously [4].Total RNA was extracted from cultured cells using RNeasy Kit (Qiagen) followed by reverse transcription using the iScript™ cDNA Synthesis Kit (Bio-Rad).Quantitative PCR was performed using Power SYBR ® Green PCR Master Mix (Applied Biosystem/Thermo Fisher Scientific, Life Technologies).β-actin levels were used as an endogenous control for normalization and data analysis was performed using the comparative threshold cycle (CT) method.The following primers were used:

Subcutaneous tumor growth
YUSIK cells were trypsinized, washed twice with ice-cold PBS and injected into 6-10 week-old nude male mice at a density of 3 x 10 5 cells in 100 µl of PBS: Matrigel (1:1 ratio) into one hind flank of nude mice (n = 5 per group).Tumor growth was measured every three days using a digital caliper and tumor volume estimated using the formula (V = L x (W 2 )/2) as described previously [4].Beginning the day before subcutaneous injection of tumor cells, mice were treated with vehicle, 10mg/kg lenvatinib, 10mg/kg CA3, or a combination of lenvatinib and CA3 at the respective doses.Mice received lenvatinib treatment daily, and CA3 treatment 3x/week.

Supplementary References
Human     Supplementary Fig. 7 Analysis of YAP expression in samples from brain metastatic melanoma patients (a-b).YAP fluorescent signal in DAPI-positive nuclei (a) and S100-positive tumor cells (b) was dichotomized into high/low groups by the associated median value and assessed in brain metastases and extracranial metastases.There were significantly more cases of brain metastases that had high YAP compared to low YAP in the DAPI compartment (a; Chi-square, p = 0.0296) and the same trend was observed in S100 compartment (b; Chi-square, p = 0.11).
(c) Examination of YAP distribution across various metastatic sites showed a prevalence of higher YAP signal in the DAPI compartment in brain metastases and low YAP signal in skin lesions, compared to all other metastatic sites (c; Chi-square, p = 0.0093 ST: Soft tissue, SK: Skin, LN: Lymph node, BR/V: Brain/Visceral, BN: Bone).The same trend was observed in the S100 compartment (d; Chi-square, p = 0.0997).(e-f): YAP expression in both the DAPI (e) and S100 compartment was compared between high or low CD34-/PAS+ staining.There were more tumors with high YAP signal among cases with high CD34-/PAS+ staining compared to cases with low CD34-/PAS+ staining (f; Chi-square, p = 0.0896).g: Two sample t-test comparing transcript levels of YAP/TAZ downstream targets in tumor samples dichotomized into high YAP or low YAP.h: Two sample t-test comparing transcript levels of CDC20, CCND1 and ANG2 in tumor samples dichotomized into high CD34-/PAS+ or low CD34-/PAS+.CL.2A  ns   Supplementary Fig. 8 Supplementary Fig. 8 The effect of YAP/TAZ inhibitors on VM junctions and tube length in patient derived and mouse melanoma brain metastasis cultures.Cl.2A (a), YUMM1.1Br(b), and B16.F10 (c) cells were treated with increasing doses of CA3 (blue) and VP (green) for 6 hours at which point VM formation was assessed and number of junctions (a-c) and meshes (d-f) were quantified.g-j: YUGANK, YUVENA, YUKRIN and YUSIK were treated with 0.5µM CA3 (blue) or 0.5µM VP (green) for 6 hours at which point VM formation was assessed, and number of junctions (g-h) and tube length (i-j) was quantified.All data are expressed as mean ± SD of at least three biological replicates and statistical significance is determined using an unpaired

1 Supplementary Fig. 2 3
Associations between vascular mimicry and metastatic site.a: Analysis of CD34+/PAS+ vessels in all intracranial and extracranial spots included in our TMA.b: Analysis of CD34-/PAS+ in all intracranial and extracranial spots included in our TMA.c-d: Analysis of CD34-/PAS+ and CD34+/PAS+ vessels in all intracranial tumor spots (75 spots total) (c) and in extracranial tumor spots (117 spots total) (d).The black line connects values from the same tumor.Significance for panels a-b assessed by unpaired T-test.****p<0.0001 and significance for panels c-d assessed by paired-Student's t-test.****p<0.0001.CD34-/PAS+ Avg.no.lesions per spot (All Cases) ✱✱✱✱ Intracranial The association of tumor volume, edema, and vascular mimicry in melanoma brain metastasis.a: Chi-squared analysis comparing tumor volume with VM (a) or BV (b) in brain metastatic samples (p=0.0146 and p=0.219 respectively).c: Linear regression of continuous tumor volume scores and continuous edema scores.Edema and tumor volume have a significant positive correlation (r 2 = 0.362).R2= 0.362 Edema Prob>|t| = 0.0009 Vascular mimicry in YUMM1.7 variants.a: Representative images of VM formation in YUMM1.7 and YUMMER1.7 cells at 6 hours in full media taken at 10x magnification.Quantification of the number of meshes (b), junctions (c), and tube length (d) in YUMM1.7 and YUMMER1.7 cells using the representative images.e: Vascular mimicry in mouse normal endothelial cells (NEC) and mouse tumor endothelial cells (TEC, bEnd.5).fh: Quantification of the number of meshes (f), junctions (g), and tube length (h).All data are expressed as mean ± SD of three biological replicates and statistical significance is determined using an unpaired Student's T-test.i-j: Representative western blot of phospho-YAP, phospho-TAZ, YAP and TAZ expression in a panel of human melanoma cell lines with quantifications of pYAP and pTAZ.Supplementary Fig. 4 Paracrine induction of VM in vitro.Quantification of the number of meshes, number of junctions, and tube length (pixels) followed by representative images (10x magnification) in non-cerebrotropic cell lines (YUKOLI (a-c), YUKSI (d-f), and YUCOT (g-i)) cultured in conditioned media from Cl.2A, YUSIK, or YUVENA for 4 hours.All experiments were completed under serum starved conditions.All data are expressed as mean ± SD of three biological replicates and statistical significance is determined using an unpaired Student's T-test.*p<0.05,**p<0.01,***p<0.001****p<0.0001Endothelial cell induction of cancer cell VM in vitro.Mouse melanoma cells (B16 and B16.F10) were indirectly co-cultured with mouse endothelial cells bEND.3 and MS1.a-c: Quantification of the number of meshes, number of junctions, and tube length (pixels) of B16 and B16.F10 cells.d: Representative images (10x magnification) in B16 and B16.F10 cells under co-culture conditions after 24-hours.All data are expressed as mean ± SD of two biological replicates and statistical significance is determined using an unpaired Student's T-test.*p<0.05,**p<0.01,***p<0.001****p<0.0001A l o n e b E n d .
Knockdown of YAP and TAZ reduce VM in vitro.Cells were transfected with scrambled control siRNA, or siRNA against YAP, TAZ, or the combination 48-hours before cells were harvested and plated on Matrigel for the VM assay (see Materials and Methods).VM was completed in serum starved media with 2% FBS.Western blot of Cl.2A (a) and YUSIK (f) depicting YAP and TAZ knockdown with siRNA after 48 hours.Quantification of the number of meshes, number of junctions, and tube length in Cl.2A (b-d) and YUSIK (g-i) followed by representative images (Cl.2A(e) and YUSIK (j) (10x magnification)) after 4-hours.All data are expressed as mean ± SD of three biological replicates and statistical significance is determined using an unpaired Student's T-test.*p<0.05,**p<0.01,***p<0.001****p<0.0001.

CTRL
Student's T-test at each time point.****p<0.0001,***p<0.001,**p<0.01,*p<0.05.Cell viability at 6-hours after drug treatment.Cell viability after treatment with CA3 (a-b) or Verteporfin (c-d) at specific doses which inhibit VM in each cell line.Raw relative luminescent units (RLU) values are presented from the DMSO treatment groups (black) and the drug treatment group (blue or green).All data are expressed as mean ± SD of at least three biological replicates and statistical significance is determined using an unpaired Student's T-test at each time point.NS: no significance.The effect of CA3 and Verteporfin on YAP/TAZ signaling.a: Western blot (left) and quantification (right) of protein expression in Cl.2A after 1µM DMSO, CA3, or verteporfin treatment in serum starved media for 6 hours.b: Western blot (left) and quantification (right) of protein expression in YUMM1.1Br after 4µM DMSO, CA3, or verteporfin treatment in full media for 6 hours.c: Western blot (left) and quantification (right) of protein expression in B16.F10 after 1µM treatment of DMSO or CA3 or with 0.5 µM DMSO or verteporfin in serum starved media for 6 hours.Western blot (left) and quantification (right) of protein expression in YUVENA (d), YUGANK (e), YUKRIN (f) and YUSIK (g) after treatment with 0.5 µM DMSO, CA3, or verteporfin in serum starved media for 6 hours.For all quantifications, treatment groups were normalized to DMSO.Total-YAP and total-TAZ levels were determined by dividing by the loading control.Western blots are representative of three independent experiments and quantifications are for the western blot shown.The effect of CA3 and Verteporfin on YAP/TAZ target genes a: Gene expression in Cl.2A after 1µM DMSO, CA3, or verteporfin treatment in serum starved media for 6 hours.b: Gene expression in YUMM1.1Br after 4µM DMSO, CA3, or verteporfin treatment in full media for 6 hours.c: Gene expression in B16.F10 after 1µM treatment of DMSO or CA3 or with 0.5 mM DMSO or verteporfin in serum starved media for 6-hours.Gene expression in YUVENA (d), YUGANK (e), YUKRIN (f) and YUSIK (g) after treatment with 0.5 µM DMSO, CA3, or verteporfin in serum starved media for 6 hours.Treatment groups were normalized to DMSO.All data are expressed as mean ± SD of at least two biological replicates and statistical significance is determined using an unpaired Student's T-test.Only significance is shown.*p<0.05,**p<0.01,***p<0.001,****p<0.0001.The effect of TED-347 in vitro, CA3 mouse weight, and TED treatment in vivo.a: Representative images of VM formation in Cl.2A cells 6 hours after DMSO or 1µM TED-347 treatment at 10x magnification.Quantification of the number of meshes (b), number of junctions (c), and the total tube length (d) at three doses.All data are expressed as mean ± SD of three biological replicates and statistical significance is determined using an unpaired Student's T-test.e: Weight of mice treated with CA3 f-h: Kaplan-Meier curves for mice receiving vehicle (n=5) or 20mg/kg TED-347 treatment (n=9) in a left ventricle injection murine model of brain metastasis.KM curves demonstrating the correlation between TED-347 treatment and overall survival (f), the time to death from first metastasis diagnosis (g), and from brain metastasis diagnosis (h).i: Representative images of immunohistochemical staining for CD34 and PAS in Cl.2A brain tumors developed after left-ventricle injection in nude mice treated as controls, or with 20mg/kg TED-347.Black arrows point to BV, red arrows point to VM structures.Scale bars represent 50µM.Quantification of BV (j; CD34+/PAS+) and VM (k; CD34-/PAS+) 15 areas from at least 2 tumors from each treatment.Students t-test was used to assess significance between vascular density in control and TED-347 treated mice.*p<0.05